The long-term objective of this proposed work is to establish a framework for using anti-peptide antibodies as probes of protein folding and dynamics. It is necessary first to investigate the mechanism of cross-reaction between intact proteins and anti-peptide antibodies. It is also necessary to develop a convenient method for studying the structures of proteins while they are bound to anti-peptide antibodies. We will study bovine pancreatic trypsin inhibitor (BPTI), a protein that has been well-characterized by biophysical and structural methods. Difference circular dichroism (CD) spectroscopy will be used to investigate the mechanism of cross-reaction at low resolution. The kinetics and thermodynamics of cross-reaction will be characterized. We will also determine if there is a correlation between the ability of peptides to fold, as judged by CD and nuclear magnetic resonance (NMR), and the ability of peptides to elicit cross-reacting antibodies. Amide proton exchange, combined with two-dimensional NMR spectroscopy (2D-NMR) will be used to study the structure of BPTI, while it is bound to anti-peptide antibodies. Hydrogen/deuterium exchange in the BPTI-antibody complex will be allowed to occur. Then, BPTI will be separated from antibody, and 2D-NMR will be used to quantitate the extent of exchange for individual amide protons in BPTI.